Name: an ex vivo method for time-lapse imaging of cultured rat mesenteric microvascular networks
Uploaded: 2017-02-09T14:00:00
Description: Watch this Scientific Journal Video about An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks at JoVE.com

This work was supported by National Institutes of Health Grant 5-P20GM103629 to WLM and the Tulane Center for Aging. We would like to thank Matthew Nice for his help with editing the protocol text.

All animal experiments and procedures were approved by the Tulane University&#39;s Institutional Animal Care and Use Committee (IACUC).
1. Surgical Procedure Setup
<ol>
	<li>Autoclave instruments, surgical supplies, and culture supplies prior to surgery. Surgical supplies for each rat include: 1 drape, 1 drape with pre-cut hole (0.5 in x 1.5 in) in the center, gauze pads, and 1 absorbent underpad. Surgical instruments include: 1 scalpel with a number 10 blade, 2 pairs of tweezers, and a pair...

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+model+for+anti-angiogenic+drug+testing+on+intact+microvascular+networks.">An ex vivo model for anti-angiogenic drug testing on intact microvascular networks.</a> PLoS One. 10 (3), e0119227 (2015).</li><li>Nicosia, R. F., Ottinetti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+microvessels+in+serum-free+matrix+culture+of+rat+aorta.+A+quantitative+assay+of+angiogenesis+in+vitro.">Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro.</a> Lab Invest. 63 (1), 115-122 (1990).</li><li>Hutter-Schmid, B., Kniewallner, K. M., Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures+as+a+model+to+study+angiogenesis+of+brain+vessels.">Organotypic brain slice cultures as a model to study angiogenesis of brain vessels.</a> Front Cell Dev Biol. 3, 52 (2015).</li><li>Sawamiphak, S., Ritter, M., Acker-Palmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+retinal+explant+cultures+to+study+ex+vivo+tip+endothelial+cell+responses.">Preparation of retinal explant cultures to study ex vivo tip endothelial cell responses.</a> Nat Protoc. 5 (10), 1659-1665 (2010).</li><li>Unoki, N., Murakami, T., Ogino, K., Nukada, M., Yoshimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+imaging+of+retinal+angiogenesis+reveals+decreased+development+and+progression+of+neovascular+sprouting+by+anecortave+desacetate.">Time-lapse imaging of retinal angiogenesis reveals decreased development and progression of neovascular sprouting by anecortave desacetate.</a> Invest Ophthalmol Vis Sci. 51 (5), 2347-2355 (2010).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+models+of+angiogenesis.">In vivo models of angiogenesis.</a> J Cell Mol Med. 10 (3), 588-612 (2006).</li><li>Kelly-Goss, M. R., Sweat, R. S., Azimi, M. S., Murfee, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+islands+during+microvascular+regression+and+regrowth+in+adult+networks.">Vascular islands during microvascular regression and regrowth in adult networks.</a> Front Physiol. 4, 108 (2013).</li><li>Kelly-Goss, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+proliferation+along+vascular+islands+during+microvascular+network+growth.">Cell proliferation along vascular islands during microvascular network growth.</a> BMC Physiol. 12, 7 (2012).</li><li>Skalak, T. C., Price, R. 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This protocol documents a method for using the rat mesentery culture model as an ex vivo tool for time-lapse imaging of microvascular network growth. Previous work in our laboratory has established the use of our model for 1) angiogenesis8, 2) lymphangiogenesis8, 3) pericyte-endothelial cell interactions8, and 4) anti-angiogenic drug testing9. The ability for imaging cultured rat mesentery tissues at multiple time poi...

Microvascular network growth and remodeling are common denominators for tissue function, wound healing, and multiple pathologies and a key process&#160;is angiogenesis, defined as the growth of new blood vessels from existing ones1,2. For tissue engineering new vessels or designing angiogenic based therapies, understanding the importance of the cellular dynamics involved in angiogenesis is critical. However, this process is complex. It can vary at specific locations within a microvascular network and involves multiple cell types (i.e. endothelial cells, smooth muscle cells, pericytes, macrophages, stem cells) and multiple systems (lymphatic networks and neural networks). Although in vitro models have contributed tremendously to examining the relationship between different cells involved in angiogenesis3, their physiological relevance can be undermined due to their limited complexity and the fact that they do not closely reflect an in vivo scenario. To overcome these limitations, three-dimensional culture systems3, ex vivo tissue models4, microfluidic systems5,6, and computational models7 have been developed and introduced in recent years. However, there is still a need for a model with time-lapse capability to investigate angiogenesis in intact microvascular networks ex vivo. The establishment of new time-lapse models for angiogenesis studies with that level of complexity will provide an invaluable tool to understand the underlying mechanisms regulating angiogenesis and to improve therapies.
A potential model that enables the ex vivo investigation of angiogenesis across an intact microvascular network is the rat mesentery culture model8. In recent work, we have demonstrated that blood and lymphatic microvascular networks remain viable after culture. More importantly, the rat mesentery culture model can be used to investigate functional pericyte-endothelial cell interactions, blood and lymphatic endothelial cell connections, and time-lapse imaging. The objective of this paper is to provide our protocol for the time-lapse imaging method. Our representative results document the multiple cell types that remain viable after the stimulation of angiogenesis with serum and offer examples of using this method for quantifying tissue specific angiogenic responses as well as endothelial cell tracking studies.

<table border="0" cellpadding="0" cellspacing="0" width="679">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:165px;">Drape</td>
			<td style="width:183px;">Cardinal Health</td>
			<td style="width:132px;">4012</td>
			<td style="width:199px;">12&#8221;x12&#8221; Bio-Shield Regular Sterilization Wraps</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Scalpel Handle</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-9843</td>
			<td style="width:199px;">Scalpel Handle, #3; Solid; 4&#34; Length</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Surgical Blade</td>
			<td>Cincinnati Surgical</td>
			<td>0110</td>
			<td>Stainless Steel; Size 10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture Dish (60mm)</td>
			<td>Thermo Scientific</td>
			<td>130181</td>
			<td>10/Sleeve</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:165px;">Graefe Forcep (curved tweezers)</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5135</td>
			<td style="width:199px;">Micro Dissecting Forceps; Serrated; Slight Curve; 0.8mm Tip Width; 4&#34; Length</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:165px;">Graefe Forcep (straight tweezers)</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5130</td>
			<td style="width:199px;">Micro Dissecting Forceps; Serrated, Straight; 0.8mm Tip Width; 4&#34; Length</td>
		</tr>
		<tr height="86">
			<td height="86" style="height:86px;width:165px;">Noyes Micro Scissor</td>
			<td style="width:183px;">Roboz Surgical Instrument</td>
			<td style="width:132px;">RS-5677</td>
			<td style="width:199px;">Noyes Micro Dissecting Spring Scissors; 
			Straight, Sharp-Blunt Points; 13mm Cutting Edge; 0.25mm Tip Width, 4 1/2&#34; Overall Length</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Gauze Pads</td>
			<td>FisherBrand</td>
			<td>13-761-52</td>
			<td style="width:199px;">Non-Sterile Cotton Gauze Sponges; 4&#34;x4&#34; 12-Ply</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Cotton-Tippled Applicators</td>
			<td>FisherBrand</td>
			<td>23-400-124</td>
			<td style="width:199px;">6&#34; Length; Wooden Shaft; Single Use Only</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">6-Well Plate</td>
			<td>Fisher Scientific</td>
			<td>08-772-49</td>
			<td style="width:199px;">Flat Bottom with Low Evaporation Lid; Polystyrene; Non-Pyrogenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Syring 5ml</td>
			<td>Fisher Scientific</td>
			<td>14-829-45</td>
			<td>Luer-Lok Tip</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Sterile Bowl</td>
			<td>Medical Action Industries Inc.</td>
			<td>01232</td>
			<td style="width:199px;">32 oz. Peel Pouch; Blue; Sterile Single Use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:165px;">6-Well Plate Inserts (CellCrown Inserts)</td>
			<td>SIGMA</td>
			<td>Z681792-3EA</td>
			<td>6-Well Plate Inserts; Non-Sterile</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:165px;">Polycarbonate Filter Membrane</td>
			<td>SIGMA</td>
			<td>TMTP04700</td>
			<td style="width:199px;">Isopore Membrane Filter; Polycarbonate; Hydrophilic; 5.0&#160;&#181;m, 47&#160;mm, White Plain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Beuthanasia</td>
			<td style="width:183px;">Schering-Plough Animal Health Corp. Union (Ordered from MWI Veterinary Supply)</td>
			<td>MWI #: 011168</td>
			<td style="width:199px;">Active Ingredient: Per 100mL, 390 mg pentobarbital sodium, 50mg phenytoin sodium&#160;</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Ketamine</td>
			<td style="width:183px;">Fort Dodge Animal Health (Ordered from MWI Veterinary Supply)</td>
			<td>MWI #: 000680</td>
			<td>Kateset 100 mg/ml</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Xylazine</td>
			<td style="width:183px;">LLOYD. Inc. (Ordered from MWI Veterinary Supply)</td>
			<td>MWI #: 000680</td>
			<td>Anased 100 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline</td>
			<td style="width:183px;">Baxter</td>
			<td>2F7122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Invitrogen</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM</td>
			<td>Invitrogen</td>
			<td>11095080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PenStrep</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Jackson ImmunoResearch</td>
			<td>001-000-162</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Saponin&#160;</td>
			<td>SIGMA</td>
			<td>S7900-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Isopropyl Alcohol</td>
			<td>Fisher Scientific</td>
			<td>S25372</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Povidone-Iodine</td>
			<td>Operand</td>
			<td>82-226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Hydrochloric Acid</td>
			<td>SIGMA</td>
			<td>320331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Methanol</td>
			<td>Fisher Scientific</td>
			<td>67-56-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Glycerol</td>
			<td>Fisher Scientific</td>
			<td>56-81-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">FITC-conjugated Lectin</td>
			<td>SIGMA</td>
			<td>L9381-2MG</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:165px;">Anti-NG2 Chondroitin Sulfate Proteoglycan Antibody</td>
			<td>SIGMA</td>
			<td>AB5320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PECAM (CD31) Antibody</td>
			<td>BD Biosciences</td>
			<td>555026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LYVE-1 Antibody</td>
			<td>AngioBio Co.</td>
			<td>11-034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rabbit Cy2-conjugated Antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-585-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Mouse Cy3-conjugated Antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-227-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Cy3-conjugated Antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>016-160-084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Live/Dead Viability/Cytotoxicity Kit</td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Goat Serum&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-Bromo-2&#39;-Deoxyuridine</td>
			<td>SIGMA</td>
			<td>B5002</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:165px;">Monoclonal Mouse Anti-Bromodeoxyuridine&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Clone Bu20a</td>
			<td>Dako</td>
			<td style="width:132px;">M074401-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Rat CD11b&#160;</td>
			<td>AbD Serotec</td>
			<td>MCA275R</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo method for time-lapse imaging of cultured rat mesenteric microvascular networks

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune cells, and the coordination with lymphatic vessels and nerves. The multi-cell, multi-system interactions necessitate the investigation of angiogenesis in a physiologically relevant environment. Thus, while the use of in vitro cell-culture models have provided mechanistic insights, a common critique is that they do not recapitulate the complexity associated with a microvascular network. The objective of this protocol is to demonstrate the ability to make time-lapse comparisons of intact microvascular networks before and after angiogenesis stimulation in cultured rat mesentery tissues. Cultured tissues contain microvascular networks that maintain their hierarchy. Immunohistochemical labeling confirms the presence of endothelial cells, smooth muscle cells, pericytes, blood vessels and lymphatic vessels. In addition, labeling tissues with BSI-lectin enables time-lapse comparison of local network regions before and after serum or growth factor stimulation characterized by increased capillary sprouting and vessel density. In comparison to common cell culture models, this method provides a tool for endothelial cell lineage studies and tissue specific angiogenic drug evaluation in physiologically relevant microvascular networks.

After 3 days in culture, tissues were labeled with a live/dead viability/cytotoxicity kit to demonstrate the viability of the microvasculature in the rat mesentery culture model (Figure 2A). The majority of cells present in the mesentery remained viable in the culture where endothelial cells were identified based on their location in microvascular segments. Endothelial cell proliferation was also confirmed by lectin/BrdU labeling (Figure 2D). Smooth muscl...

Watch this Scientific Journal Video about An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks at JoVE.com

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

The overall goal of this experiment is to demonstrate the use of the rat mesenteric culture model for the time-lapse investigation of microvascular network re-modeling ex vivo to evaluate parasite endothelial cell interactions, blood lymphatic endothelial cell connections, or anti-angiogenic drug effects. This method can help answer key questions in the field of microvascular remodeling, including how do microvasculars know where to grow, and what is the fate of endothelial cell segments. The main advantage of this technique is that real, intact microvascular networks can be imaged before and after growth ex vivo.Demonstrating the procedure will be Sadegh Azimi, a graduate student in our laboratory who developed the method. Begin by placing an ethanol-sterilized plexiglass platform on top of a sterile, absorbent under-pad and an anesthetized adult male rat on top of the platform. Disinfect the shaved abdominal region of the rat with two 70%isopropyl alcohol wipes, followed by povidone-iodine.Starting one inch below the sternum, use a scalpel to make a three-quarter to one and one-quarter inch incision in the gut through the skin, connective tissue, and muscle. Then, place a drape with a precut hole over the animal and place a surgical stage over the drape, taking care to align the openings in both materials with the incision. Using sterile cotton-tipped applicators, pull the ileum through the surgical stage opening along with six to eight mesenteric windows.Use a sterile syringe to drip 37 degrees Celsius sterile saline onto the exposed tissues as needed. When all of the windows have been externalized, use tweezers to grasp a fat pad and use scissors to harvest the windows, leaving a two millimeter fat border around each mesenteric tissue. Take care that the tweezers do not touch the mesenteric windows by only touching the surrounding fat borders.Wash each window one time in 37 degree Celsius PBS and one time in 37 degree Celsius medium as it is collected. Returning the ileum to the abdominal cavity after all of the windows have been isolated. In a sterile laminar flow hood, use tweezers to transfer each mesenteric tissue by the fat border onto the polycarbonate filter membrane of a cell culture plate insert.Use the fat pad to quickly spread the tissue samples, taking care not to touch the windows. Then, invert the insert and place into one well of a six well plate so the tissue lays flat on the bottom of the well. Cover the tissue with three millimeters of medium, and transfer the remaining windows to the plate in the same manner.Then, culture the mesentery windows under standard tissue culture incubator conditions for up to five days. On the day of imaging, supplement the cultures with conjugated BSI lectin and return the plate to the incubator. After 30 minutes, wash the samples two times with lectin-free medium and transfer the plate to a fluorescent microscope stage.Identify the blood and lymphatic vessels based on their morphology and network structures. Then, locate a network region of interest on each tissue to obtain images, taking note of the location to ensure that the same region is captured in subsequent images. When all of the images have been obtained, return the samples to the incubator until the next imaging session or the appropriate experimental endpoint.After three days in culture, the majority of the cells present in the mesentery are viable, with the presence of the endothelial cells confirmed by their location in the microvascular segments. Endothelial cell proliferation can also be confirmed by lectin/BrdU labeling, while the presence of smooth muscle cells and parasites along the vessels can be confirmed with NG2 labeling. Branching lymphatic and blood microvascular network labeling further confirms the maintenance of the lymphatic cell phenotype in the cultured tissue.Supplementation of the mesentery window tissue culture medium with 10%serum for three days induces a robust angiogenic response with new vessel segments and capillary sprouts identified after five days of stimulation. The time lapse imaging allows quantification of the number of vessels per vascular area and the number of capillary sprouts per vascular area from one four x image per tissue. Time lapse comparisons of network regions also enable the tracking of endothelial cell segments and the identification of blood and lymphatic vessel mis-patterning.Further, lectin and Cd11b labeling additionally confirms the presence of interstitial resident macrophages in the remodeling networks. Once mastered, this technique should be able to be completed in under four hours. After watching this video, you should have a good understanding of how to harvest, culture, and time-lapse image rat mesenteric tissues.

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Research

JoVE Journal

Developmental Biology

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch&#039;s Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

Ex Vivo Perfusion of the Rodent Placenta

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An assay is described here to perfuse an isolated rat placenta and evaluate the maternal-to-fetal translocation of xenobiotics ex vivo. In addition, the evaluation of physiological processes such as fluid flow to the fetus and placental metabolism may be conducted with this methodology. This technique is suitable for evaluating maternal-to-fetal kinetics of pharmaceutical candidates or environmental contaminants. In contrast to current alternative approaches, this methodology allows the evaluation of the isolated maternal-fetal vasculature, with the systemic neural or immune involvement removed, allowing any observed changes in physiological function to be attributable to local factors within the isolated tissue.

Here is presented a protocol of ex vivo maternal-fetal vascular perfusion to enable the administration of a test article into maternal vasculature and to evaluate placental transfer of xenobiotic particles or pharmacological agents in addition to alterations in placental physiology.

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...